Proteolytic enzyme preparations whose substrate specificity requirements are broad can be utilized to digest proteins containing tyrosine-nucleotide phosphodiester residues yielding free tyrosine O-nucleotide. Pronase and proteinase K digests of [32P,3H] adenylylated glutamine synthetase yielded tyrosine O-AMP which migrated as a single spot on thin layer (silica) chromatography, and chromatographed as a discrete peak on reverse phase high pressure liquid chromatography. Confirmation that the isolated radiolabeled material was a phosphodiester was achieved by snake venom exonuclease treatment which resulted in the formation of [3H,32P]AMP. These results suggest that protease treatment of proteins containing putative tyrosine O-nucleotide residues will yield the free modified tyrosine which can be isolated by high pressure chromatography prior to treatment with snake venom exonuclease for nucleotide identification.